Nitric Oxide (NO) regulates cardiovascular homeostasis by binding to soluble guanylate cyclase (sGC), leading to cGMP production, reduced cytosolic calcium concentration ([Ca²⁺]ᵢ) and vasorelaxation. Thrombospondin-1 (TSP-1), a secreted matricellular protein, was recently discovered to inhibit NO signaling and sGC activity. Inhibition of sGC requires binding to cell-surface receptor CD47. Here, I show that a TSP-1 C-terminal fragment (E3CaG1) readily inhibits sGC in Jurkat T cells, and that inhibition requires an increase in [Ca²⁺]ᵢ. Using digital imaging microscopy on live cells, I further show that E3CaG1 binding results in a substantial increase in [Ca²⁺]ᵢ, up to 300 nM. Addition of angiotensin II, a potent vasoconstrictor known to increase [Ca²⁺]ᵢ, also strongly inhibits sGC activity. sGC isolated from calcium-treated cells or from cell-free lysates supplemented with Ca²⁺ remains inhibited, while addition of kinase inhibitors staurosporine, genistein, PP1 or PP2 reverse inhibition, indicating inhibition likely involves a tyrosine kinase, more specifically, a src family kinase. Rat sGC is also inhibited by lysates supplemented with Ca²⁺, suggesting that the site of modification is at an evolutionarily conserved residue. Inhibition is through an increase in K(m) for GTP, which rises to 834 μM for the NO-stimulated protein, a 13-fold increase over the uninhibited protein. Compounds YC-1 and BAY 41-2272, allosteric stimulators of sGC that are of interest for treating hypertension, overcome E3CaG1-mediated inhibition of NO-ligated sGC. Taken together, these data suggest that sGC not only lowers [Ca²⁺]ᵢ in response to NO, inducing vasodilation, but is also inhibited by high [Ca²⁺]ᵢ, providing a fine balance between signals for vasodilation and vasoconstriction.

Nitric Oxide (NO) regulates cardiovascular homeostasis by binding to soluble guanylate cyclase (sGC), leading to cGMP production, reduced cytosolic calcium concentration ([Ca²⁺]ᵢ) and vasorelaxation. Thrombospondin-1 (TSP-1), a secreted matricellular protein, was recently discovered to inhibit NO signaling and sGC activity. Inhibition of sGC requires binding to cell-surface receptor CD47. Here, I show that a TSP-1 C-terminal fragment (E3CaG1) readily inhibits sGC in Jurkat T cells, and that inhibition requires an increase in [Ca²⁺]ᵢ. Using digital imaging microscopy on live cells, I further show that E3CaG1 binding results in a substantial increase in [Ca²⁺]ᵢ, up to 300 nM. Addition of angiotensin II, a potent vasoconstrictor known to increase [Ca²⁺]ᵢ, also strongly inhibits sGC activity. sGC isolated from calcium-treated cells or from cell-free lysates supplemented with Ca²⁺ remains inhibited, while addition of kinase inhibitors staurosporine, genistein, PP1 or PP2 reverse inhibition, indicating inhibition likely involves a tyrosine kinase, more specifically, a src family kinase. Rat sGC is also inhibited by lysates supplemented with Ca²⁺, suggesting that the site of modification is at an evolutionarily conserved residue. Inhibition is through an increase in K(m) for GTP, which rises to 834 μM for the NO-stimulated protein, a 13-fold increase over the uninhibited protein. Compounds YC-1 and BAY 41-2272, allosteric stimulators of sGC that are of interest for treating hypertension, overcome E3CaG1-mediated inhibition of NO-ligated sGC. Taken together, these data suggest that sGC not only lowers [Ca²⁺]ᵢ in response to NO, inducing vasodilation, but is also inhibited by high [Ca²⁺]ᵢ, providing a fine balance between signals for vasodilation and vasoconstriction.

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dc.type

text

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dc.type

Electronic Dissertation

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dc.subject

Molecular & Cellular Biology

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thesis.degree.name

Ph.D.

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thesis.degree.level

doctoral

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thesis.degree.discipline

Graduate College

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thesis.degree.discipline

Molecular & Cellular Biology

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thesis.degree.grantor

University of Arizona

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dc.contributor.advisor

Montfort, William R.

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dc.contributor.committeemember

Camenisch, Todd D.

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dc.contributor.committeemember

Miesfeld, Roger L.

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dc.contributor.committeemember

Tsao, Tsu-Shuen

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dc.contributor.committeemember

Weinert, Ted A.

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dc.contributor.committeemember

Montfort, William R.

en_US

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